Therapeutic agent for phosphodiesterase inhibition and its related disorders

ABSTRACT

The pharmaceutical composition for treatment of phosphodiesterase related disorders comprises of administering to the subject a therapeutically effective amount of a compound of Formula-I, or compounds selected from the group consisting of Niclosamide, Oxyclozanide, Rafoxanide, Closantel, Dibromsalan, Metabromsalan. Tribromsalan and Nitazoxanide, and prodrugs, metabolites, or pharmaceutically acceptable salts, solvates and polymorphs thereof in a pharmaceutically acceptable carrier, vehicle, or diluents.

FIELD OF THE INVENTION

The present disclosure relates to treatment of phosphodiesterase (PDE)related disorders.

BACKGROUND OF THE INVENTION

A phosphodiesterase is an enzyme class that breaks a phosphodiesterbond. The cyclic nucleotide phosphodiesterases (PDE) comprise a group ofenzymes that degrade the phosphodiester bond in the second messengermolecules namely cAMP (cyclic adenosine monophosphate) and cGMP (cyclicguanosine monophosphate). They regulate the localization, duration, andamplitude of cyclic nucleotide signaling within subcellular domains.PDEs are therefore important regulators of signal transduction mediatedby these second messenger molecules. PDEs are also downstream effectorsof cAMP and cGMP. PDEs are closely related to the regulation of eachspecific transduction signal, and therefore multiple PDEs play importantroles in modulating each cellular function.

The PDE superfamily of enzymes is classified into 11 families, namelyPDE1 to PDE11, in mammals. Different PDEs of the same family arefunctionally related despite the fact that their amino acid sequencescan show considerable divergence. PDEs have different substratespecificities. Some are cAMP selective hydrolases (PDE4, PDE7 and PDE8),while others are cGMP selective (PDE5, PDE6 and PDE9). Others canhydrolyze both cAMP and cGMP (PDE1, PDE2, PDE3, PDE10 and PDE11). Thereis evidence that many of these PDEs are tightly connected to differentphysiological functions in the body and hence by inference also todifferent pathological conditions. Another significant factor in theregulation of PDEs and their role in the cyclic nucleotide metabolism isthe fact that PDE isoenzymes and families tend to be verytissue-specific.

PDE enzymes are often targets for pharmacological inhibition due totheir unique tissue distribution, structural properties, and functionalproperties. Inhibitors of PDE can prolong or enhance the effects ofphysiological processes mediated by cAMP or cGMP by inhibition of theirdegradation by PDE. Selective and non-selective PDE inhibitors have beenidentified as new potential therapeutics for several diseases. SelectivePDE inhibitors are being used as well as investigated in a wide range ofdiseases including:

(i) PDE1 inhibitor Vinpocetine has been investigated in clinical trialsfor its neuroprotective effects. Vinpocetine also has antioxidanteffects and antiulcer activity.

(ii) PDE2 inhibitors have been explored for their use in sepsis,cognitive disorders and Acute Respiratory Distress Syndrome (ARDS).

(iii) PDE3 inhibitors are used for intermittent claudication andproposed for use in Airways disease and fertility (male and femalecontraceptives). PDE3 inhibitor also has shown anti-asthmatic effects byboth anti-inflammatory and bronchodilator properties. In the treatmentof atopic dermatitis, the mixed PDE3-PDE4 inhibitor Zardaverine was amore potent inhibitor of T cell proliferation than Rolipram, a selectivePDE4 inhibitor.(iv) PDE4 inhibitors have shown potential to treat asthma, COPD,allergic rhinitis, psoriasis, multiple sclerosis, depression,Alzheimer's disease, memory loss, cancer, dermatitis and schizophrenia.Pentoxifylline, a PDE4 inhibitor, has the potential to enhancecirculation and may have applicability in treatment of diabetes,fibrotic disorders, peripheral nerve damage, and microvascular injuries.PDE4 inhibitors have also shown potential in the treatment of AIDS,arthritis like rheumatoid-, psoriatic- and osteo-arthritis, bronchitis,endotoxin shock, inflammatory bowel diseases like ulcerative colitis andCrohn's disease, adult respiratory distress syndrome (ARDS), allergicconjunctivitis, dry eye disorder and other inflammatory diseases. PDE4inhibitors suppress the release of cytokines and other inflammatorysignals, and inhibit the production of reactive oxygen species.Crisaborole is a nonsteroidal, topically administered, boron-containing,anti-inflammatory compound that inhibits PDE4 activity and therebysuppresses the release of TNF-α, IL-12, IL-23 and other cytokines.Crisaborole is approved for the treatment of mild-to-moderate atopicdermatitis (eczema) in patients 2 years of age and older. Phase I andphase II trials of Crisaborole in patients with psoriasis have also beencompleted. Apremilast which is also a PDE4 inhibitor has been approvedfor treatment of psoriatic arthritis and psoriasis. PDE4 inhibitors havebeen demonstrated to be beneficial in the treatment of lung inflammationand fibrosis.(v) PDE5 inhibitors have been used to treat erectile dysfunction, sexualdysfunction in females, cardiovascular disease, premature ejaculation,stroke, leukemia and renal failure. Sildenafil (PDE5 inhibitor) is alsocurrently being investigated for its myo- and cardioprotective effects,with particular interest being given to the compound's therapeutic valuein the treatment of Duchenne muscular dystrophy. PDE5 is abundantlyexpressed in lung tissue, and appears to be upregulated in pulmonaryarterial hypertension (PAH). PDE5 inhibitors Sildenafil and Tadalafilare two of the recommended first-line therapies for PAH patients inWorld Health Organization functional classes II or III.(vi) PDE7 inhibitors have been used in inflammatory diseases. PDE7Ainhibitor compound ASB16165 has been shown to inhibit T lymphocyteactivation and suppresses skin inflammation and impair proliferation ofkeratinocytes both in vitro and in vivo. Studies indicate PDE7A mightregulate TNF-α production in keratinocytes in a cAMP-dependent fashionand have reduced skin inflammation in animal model. Specific inhibitorsof PDE7 have also been reported as potential new drugs for the treatmentof neurological disorders because of their ability to increaseintracellular levels of cAMP.(vii) PDE9 inhibitors have been explored for treatment of Alzheimer'sdisease.(viii) PDE10 inhibitors have shown potential in treatment ofSchizophrenia and Huntington's disease.

Non-selective PDE inhibitors such as Caffeine, Aminophylline,Pentoxyphylline, Theophylline etc. have the potential to enhancecirculation and may have applicability in treatment of diabetes,fibrotic disorders, peripheral nerve damage, and microvascular injuriesor as bronchodilators. Thus, even nonselective phosphodiesteraseinhibitors have therapeutic utility and have been shown to inhibit TNF-αand leukotriene synthesis and reduce inflammation and innate immunity.

Inflammatory cells such as Basophils, B-lymphocytes, Dendritic cells,Endothelial cells, Eosinophils, Macrophages, Mast cells, Monocytes,Neutrophils and T-lymphocytes express various PDEs at different levelsand inhibition of PDEs in these cells can lead to anti-inflammatoryeffects in different tissues. These cells are also involved in severalimmunological functions and are involved in pathogenesis of manydiseases.

Phosphodiesterase inhibitors have shown efficacy in treatment of avariety of inflammatory skin diseases. Skin diseases such as psoriasis,dermatitis or eczema, acne, rosacea have complex pathology, with somecommon pathways involving in many of these diseases. Some of the skindiseases such as acne and dermatitis may be triggered from bacterialinfections but may also have an inflammatory component. Alternatively,inflammatory skin diseases are triggered by causes other than bacterialinfection (non-infectious origin), e.g. immune system disorder,medication, food allergy, emotional stress, UV radiation, soaps andfragrances, allergens, etc. In a group of embodiments, the compounds ofFormula I described herein are suitable for treatment of inflammatoryskin conditions such as psoriasis, rosacea or dermatitis. In anothergroup of embodiments, the compounds of Formula I described herein aresuitable for treatment of inflammatory skin conditions such as psoriasisor rosacea. In an alternate group of embodiments, the compounds ofFormula I described herein are suitable for treatment of secondaryinflammation associated with skin conditions which may be initiallytriggered by bacterial infections. In further embodiments, the compoundsof Formula I described herein are suitable for treatment of dermatitisor eczema not associated with or originating from an underlyingmicrobial infection.

Psoriasis includes, and is not limited to, plaque psoriasis (psoriasisvulgaris), guttate psoriasis, inverse psoriasis, erythrodermic psoriasisand pustular psoriasis having varied clinical signs and incidencesamongst psoriatic population. Dermatitis includes, and is not limitedto, atopic dermatitis, contact dermatitis, stasis dermatitis,dyshidrotic dermatitis, nummular dermatitis, neurodermatitis, seborrheicdermatitis and the like. Rosacea includes, and is not limited to,erythematotelangiectatic rosacea, papulopustular rosacea, phymatousrosacea, ocular rosacea and the like. Effective treatment of skindiseases like psoriasis, rosacea and dermatitis requires management ofdifferent inflammatory processes involved in the pathogenesis of thedisease.

Psoriasis is characterized by hyperplasia of epidermal keratinocytes,angiogenesis, and infiltration of T-lymphocytes, neutrophils, and othertypes of leukocyte in the affected skin. The proliferative keratinocyteresponse is thought to be due to activation of the cellular immunesystem, with T-cells, dendritic cells, and various immune-relatedcytokines and chemokines implicated in pathogenesis. Further, histamineand proteases play a role in the pathogenesis of psoriasis. Currently,psoriasis has been considered as a T-lymphocyte-mediated autoimmunedisease, and new biological therapies that target T-cells have justentered routine clinical practice as IL-17 and IL-23 monoclonalantibodies indicating importance of IL-17/IL-23 axis in pathogenesis ofpsoriasis. However, current observations suggest that although activatedT-lymphocytes have an undisputed role in the pathogenesis of psoriasis,there are other regulatory systems that contribute to the inflammatoryand proliferative processes of psoriasis. The current pathogenesis ofpsoriasis involves keratinocytes, NK T cells, plasmacytoid dendriticcells, macrophages which release several cytokines like TNF-α, IL-6,IL-1, IFN-γ leading to activation of myeloid dendritic cells whichrelease IL-23 and IL-12 to activate Th-17 and Th-1 cells. The activatedTh-17 cells release cytokines like IL-17A, IL-17F and IL-22, whereasactivated Th-1 cells release TNF-α and IFN-γ which activatekeratinocytes which in turn release several cytokines and chemokinesthereby continuing the diseases state. PDE inhibition has shown to havereduced cytokines involving IL-17/IL-23 axis. One such regulatory systeminvolves cyclic nucleotides that play key role in psoriasis. Themediator system, cAMP− cGMP as an off-on system, is not as simple asonce perceived and cAMP can either stimulate or inhibit cellproliferation. The PDE inhibitors Aminophylline and Theophylline, whichincrease cAMP levels, have been shown to be of benefit in the treatmentof psoriasis.

Increased cAMP-phosphodiesterase activity in peripheral blood leukocytesis associated with the immune and inflammatory hyper reactivity thatcharacterizes atopic dermatitis. Atopic dermatitis is responsive to avariety of enzyme inhibitors. The ability of selective high-potency PDEinhibitors to reduce prostaglandin E2, IL-10, and IL-4 production inatopic mononuclear leukocyte cultures has been demonstrated in vitro. Aclinical study of a PDE4 inhibitor in 20 patients with atopic dermatitisdemonstrated significant reductions of all inflammatory parameters. PDEinhibitors modulate several pathways contributing to the exaggeratedimmune and inflammatory responses, which characterize atopic dermatitis.Thus, PDE inhibitors may provide a useful alternative to theover-reliance on corticosteroid therapy in atopic diseases likepsoriasis, eczema and rosacea. Further, involvement of IL-23 and Th17cytokines has been described in allergic contact dermatitis which showsinvolvement of cytokines like IL-17A, IL-17F and IL-22 in the disease.

Rosacea is a chronic and potentially life-disruptive disorder primarilyof the facial skin inflammation, often characterized by flare-ups andremissions. It is observed as a redness on the cheeks, nose, chin orforehead. In some cases, rosacea may also occur on the neck, chest,scalp or ears; which is may be due to factors related to blood flow,skin bacteria, microscopic skin mites (Demodex), irritation offollicles, sun damage of the connective tissue under the skin, anabnormal immune or inflammatory response, or psychological factors.Apremilast (PDE4 inhibitor) has been investigated for the treatment ofrosacea on a small population and has shown promising results. Recentstudy shows involvement of Th1/Th17 cells in pathogenesis of allsubtypes of rosacea.

PDE inhibitors can provide alternatives for treatment of fibrotic skindiseases through various routes of administration, including topicaladministration. Sildenafil, a PDE5 inhibitor is an effective drug totreat the manifestations of scleroderma vasculopathy, such as Raynaud'sphenomenon (RP), digital ulcers (DU) and/or necroses (N), and pulmonaryhypertension (PH). Thus, both cyclic nucleotides (cAMP and cGMP)specific PDE inhibitors can provide treatment options in scleroderma.This is exemplified by use of the PDE3 inhibitor Cilostazol fortreatment of Raynaud's syndrome (RS).

Thus, PDE inhibitors can play key role in alleviation of several immuneand &in diseases. Many of these immune and skin diseases are treatedwith corticosteroids and drugs having several limitations and safetyissues. Therefore, there is a need for safe and effective compositionsfor treatment of PDE related disorders.

SUMMARY OF THE INVENTION

This disclosure is based in part on the finding that certainanti-parasitic drugs (e.g., certain anti-helminthic drugs, certainanti-protozoal drugs) inhibit phosphodiesterases. Disclosed herein isthe finding that compositions comprising compounds of Formula-I areuseful for treating phosphodiesterase related disorders in a mammal,

wherein R1 is —H or —COCH₃; R₂ is —H, —Cl, —Br, or —I; R₃ is —H, —Cl,—Br, or —I; R₄ is —H. or —Cl; Ar is

wherein R₅ is —H, —NO₂, or —Br, R₆ is —H, or —Cl; R₇ is —H, or —OH, —Cl;R₈ is —H, or —Cl.

The compounds of Formula-I are salicylamide derivatives. In oneembodiment, the compounds of Formula-I include anti-parasitic drugs suchas Niclosamide, Oxyclozanide, Rafoxanide, Closantel, Dibromsalan,Metabromsalan, Tribromsalan and Nitazoxanide. In another embodiment, thecompounds of Formula-I include anti-parasitic drugs such as Niclosamide,Oxyclozanide, Rafoxanide, Closantel, Dibromsalan, Metabromsalan, andTribromsalan. It has been found that compounds of formula I areinhibitors of phosphodiesterases and hence may provide safe andeffective treatment for PDE related disorders. Accordingly, providedherein is a method for treatment of phosphodiesterase (PDE) relateddisorders by administering to a subject in need thereof atherapeutically effective amount of compounds of Formula-I, including,for example, anti-parasitic drugs such as Niclosamide, Oxyclozanide,Rafoxanide, Closantel, Dibromsalan, Metabromsalan, Tribromsalan andNitazoxanide, and their prodrugs, metabolites, or pharmaceuticallyacceptable salts, solvates and polymorphs thereof in a pharmaceuticallyacceptable carrier, vehicle, or diluents.

The compounds of Formula-I, or prodrugs or metabolites, orpharmaceutically acceptable salts, solvates and polymorphs thereof, maybe administered in a suitable form. The mammal may be a primate, canine,feline, bovine, ovine, porcine, camelid, caprine, rodent or equine.Preferably, the primate is a human.

The PDE related disorders may be treated by administering atherapeutically effective amount of compound of Formula-I, which mayinclude, but is not limited to, Niclosamide, Oxyclozanide, Rafoxanide,Closantel, Dibromsalan, Metabromsalan, Tribromsalan and Nitazoxanide andtheir prodrugs or metabolites, or pharmaceutically acceptable salts,solvates and polymorphs thereof in a pharmaceutically acceptablecarrier, vehicle, or diluents. The administration may be conductedhourly, daily, weekly or monthly. The daily administration may involveanywhere from one to six administrations each day.

The compound of Formula-I may be administered via an oral, intravenous,intraperitoneal, ophthalmic, parenteral, topical, transdermal,subcutaneous, subdural, intravenous, intramuscular, intradermal,intrathecal, intraperitoneal, intracerebral, intraarterial,intralesional, localized or pulmonary route. When administered by oralroute, the dosage of the compound of Formula-I is preferably about 1 mgto 3000 mg daily in the form of tablet, capsule, solution, suspension,syrup or dental paste. When administered by ophthalmic route, the dosageof the compound of Formula-I is preferably at a concentration of 0.001%to 10.0% w/v administered 1 to 3 drops (i.e 0.05 mL to 0.15 mL) at eachtime up to 3 times a day. The ophthalmic composition is administered aseye drops or eye gel. When administered by a topical route, the dosageof the compound of Formula-I is preferably at a concentration of 0.001%to 20.0% w/w applied up to three times a day in a quantity that issufficient to cover the affected area by the formulation with a layer ofapproximately 0.1 mm to 5 mm thickness. The topical composition may bein the form of a cream, gel, patch, ointment, topical swab, emulsion,paste, shampoo, solution or spray applied with or without applicator.When administered by intravenous, intradermal, intralesional orsubcutaneous route the concentration of therapeutically activeingredient of compound of Formula-I is preferably about 0.001% to 10.0%w/v administered up to 3 times a day with maximum dose of 500 mg each byinjection or infusion. The pulmonary composition may be administered toa mammal as an inhaler, nebulizer or vaporizer in a concentration ofabout 0.001% to 10.0% w/w administered up to 10 times a day.

The present invention further provides a composition and methods oftreating PDE related disorders mediated by over-activation of one ormore PDE isoforms in a mammal, comprising administering atherapeutically effective amount of compound of Formula-I, a prodrugs ormetabolites, or pharmaceutically acceptable salts, solvates andpolymorphs thereof to a patient in need thereof. Preferably, thecompound of Formula-I is Niclosamide, Oxyclozanide, Rafoxanide,Closantel, Dibromsalan, Metabromsalan, Tribromsalan and Nitazoxanide.The therapeutically effective amount of the compound of Formula-I,prodrug thereof, or pharmaceutically acceptable salt or solvates thereofmay be provided in a pharmaceutically acceptable carrier, vehicle ordiluent.

Preferably, the PDE related disorders are pulmonary disorders e.g.:asthma, bronchitis, chronic obstructive pulmonary disease (COPD), adultrespiratory distress syndrome (ARDS), pulmonary arterial hypertension;allergic diseases such as e.g.: allergic rhinitis and allergicconjunctivitis; endotoxin shock; inflammatory bowel diseases e.g.ulcerative colitis and Crohn's disease; joint disorders such asarthritis, psoriatic arthritis and osteoarthritis; coronary heartdisease; intermittent claudication; mental disorders e.g.: dementia,depression, schizophrenia; erectile dysfunction; Duchenne musculardystrophy; male and female fertility; Dry eye disorder, psoriasis,rosacea, dermatitis; fibrotic skin diseases such as keloids,hypertrophic scarring, collagenoma and systemic sclerosis orscleroderma, Alopecia areata, Lichen Planus, Pemphigus foliaceus,Pemphigus vulgaris, Chronic periodontitis, Dermatitis Herpetiformis,Vitiligo and Bullous Pemphigoid. In some embodiments of this invention,Nitazoxanide in combination with either an antiviral compound or abroad-spectrum antibiotic does not include the treatment of arthritis,osteoarthritis, and bursitis. In some embodiments of this invention,Nitazoxanide alone (i.e., not in combination with either an antiviralcompound or a broad-spectrum antibiotic), is administered to a subjectin need thereof to treat arthritis, osteoarthritis, and bursitis.

DESCRIPTION OF THE INVENTION

Provided herein are compositions comprising compounds of Formula-I whichare antiparasitic drugs, their prodrugs and salts, solvates andpolymorphs as well as all possible diastereomers and all enantiomericforms as well as all combinations of diastereomers and enantiomers,including racemic mixtures and all tautomeric forms.

A subject according to the invention can be any human or animal with PDErelated disorders. The animal may be a mammal. The mammal may be acanine, feline, primate, bovine, ovine, porcine, camelia, caprine,rodent, or equine. Preferably, the primate is a human.

In one embodiment, a compound of Formula I is Niclosamide, which, inaddition to being an antihelminthic and a piscicide has also beenreported as effective in the treatment of neoplasms, protozoal andparasitic infections, acne of bacterial origin, severe acute respiratorysyndrome (SARS), Swimmers' itch, as an anti-anthrax toxin, as ananti-neoplastic, and for preventing and/or treating pulmonary fibrosis.In another embodiment, compounds of Formula I are halogenatedsalicylanilide compounds such as Closantel, Rafoxanide, Oxyclozanide,and Niclosamide, which have been used to treat skin conditions such asimpetigo, infected dermatitis (for example infected atopic dermatitis),infected eczema, infected skin wounds, infected burns and infectedulcers (for example diabetic ulcers), originating from bacterialinfections arising from Gram-positive bacteria such as Staphylococcus,in particular Staphylococcus aureus, and Streptococcus, in particularStreptococcus pyogenes. Oxyclozanide and Rafoxanide are salicylanilidesof Formula I which have been used in the treatment and control ofFascioliasis in ruminants. Bromosalans (e.g., Dibromsalan andTribromsalan) are biphenolic compounds used as fasciolicides effectiveagainst juvenile flukes. Brominated derivatives including Dibromsalan,Metabromsalan, and Tribromsalan are used as disinfectants withantibacterial and antifungal activities. Nitazoxanide, a prodrug whichupon oral administration, rapidly hydrolyzes to its active metabolite,Tizoxanide, is used for the treatment of infectious diarrhea, illnesscaused by other protozoa and/or helminths. Nitazoxanide is currently inPhase II clinical trials for the treatment of hepatitis C, incombination with peginterferon alfa-2a and ribavirin. Nitazoxanide alonehas shown preliminary evidence of efficacy in the treatment of chronichepatitis B. Recently, use of Nitazoxanide for treatment ofMycobacterium tuberculosis has been proposed. Accordingly, most knowncompounds of Formula I are generally recognized as anti-infectives(e.g., anti-bacterials, anti-parastics, anti-protozoals). Describedherein are methods for re-purposing compounds of Formula I forconditions that are not associated with infections, i.e., PDE relatedconditions.

The compounds of Formula-I include and are not limited to Niclosamide,Oxyclozanide, Rafoxanide, Closantel, Dibromsalan, Metabromsalan,Tribromsalan and Nitazoxanide, their prodrugs, their metabolites, orpharmaceutically acceptable salts, solvates and polymorphs thereof aresurprisingly found to be effective in inhibiting one or morephosphodiesterases like PDE1, PDE2, PDE3, PDE4, PDE5, PDE7, PDE9 andPDE10 and therefore can be utilized for the treatment of PDE relateddisorders. PDE related disorders associated with elevated PDE activityinclude and are not limited to pulmonary disorders e.g: asthma,bronchitis, chronic obstructive pulmonary disease (COPD), pulmonaryarterial hypertension; allergic diseases e.g: allergic rhinitis andallergic conjunctivitis; endotoxin shock; inflammatory bowel diseasese.g. ulcerative colitis and Crohn's disease; joint disorders such asarthritis, psoriatic arthritis and osteoarthritis; coronary heartdisease; intermittent claudication; mental disorders e.g.: dementia,depression, schizophrenia; erectile dysfunction; Duchenne musculardystrophy; male and female fertility; Dry eye disorder, psoriasis,rosacea, dermatitis; fibrotic skin diseases e.g. keloids, hypertrophicscarring, collagenoma and systemic sclerosis or scleroderma and others.Inhibition of PDE confers a therapeutic effect on PDE-related disordersas shown in the Examples section.

The compounds of formula I are found to inhibit release of severalcytokines from Peripheral Blood Mononuclear Cells (PBMCs), in particularTh-17 cytokines like IL-17A, IL-17F and IL22 along with inhibition ofcytokine release of IL-12 (See Example 5). These cytokines are among PDEregulated downstream cytokines. Considering this effective inhibition ofTh-17 cytokines, the compounds of formula I can be utilized fortreatment of several diseases like Alopecia areata, Lichen Planus,Pemphigus foliaceus, Pemphigus vulgaris, chronic periodontitis,Dermatitis Herpetiformis, Vitiligo and Bullous Pemphigoid.

An embodiment of this invention includes compositions of compoundshaving the structure of Formula-I for treating phosphodiesterase relateddisorders in a mammal,

wherein R1 is —H or —COCH₃; R₂ is —H, —Cl, —Br, or —I; R₃ is —H, —Cl,—Br, or —I; R₄ is —H, or —Cl; Ar is

wherein R₅ is —H, —NO₂, or —Br, R₆ is —H, or —Cl; R₇ is —H, or —OH, —Cl;R₈ is —H, or —Cl.

Also an embodiment of this invention is a method of treatingphosphodiesterase related disorders in a mammal, comprisingadministering a therapeutically effective amount of a compositioncomprising a compound of Formula-I, or a prodrug or a metabolitethereof, or pharmaceutically acceptable salts, solvates and polymorphsthereof in a pharmaceutically acceptable carrier, vehicle, or diluentsto a subject in need of such treatment.

In an aspect, provided is a method for inhibiting a phosphodiesterase,comprising contacting a phosphodiesterase with a compound of formula-I

wherein R₁ is —H or —COCH₃; R₂ is —H, —Cl, —Br, or —I; R₃ is —H, —Cl,—Br, or —I; R₄ is —H, or —Cl; Ar is

wherein R₅ is —H, —NO₂, or —Br,R₆ is —H, or —Cl; R₇ is —H, —OH, or —Cl; R₈ is —H, or —Cl; andenantiomers, diastereomers, prodrugs or metabolites thereof, orpharmaceutically acceptable salts, solvates and polymorphs thereof.

Definitions

The term “therapeutically effective amount” refers to the amountrequired for reduction in the severity of symptoms of the PDE relateddisorders.

Psoriasis may include types of psoriasis like Plaque psoriasis(psoriasis vulgaris), Guttate psoriasis, Inverse psoriasis,Erythrodermic psoriasis and Pustular Psoriasis. The psoriasis may beexpressed at one or more body organs simultaneously including scalp.

Dermatitis may include various types of dermatitis like atopicdermatitis, contact dermatitis, stasis dermatitis, DyshidroticDermatitis, Nummular Dermatitis, Neurodermatitis and SeborrheicDermatitis.

Rosacea may include Erythematotelangiectatic Rosacea, PapulopustularRosacea, Phymatous Rosacea and Ocular Rosacea.

Methods of Administration

One aspect of the disclosure contemplates the use of therapeuticallyeffective amount of anti-parasitic drugs of Formula-I such asNiclosamide, Oxyclozanide, Rafoxanide, Closantel, Dibromsalan,Metabromsalan, Tribromsalan and Nitazoxanide, their prodrugs, theirmetabolites, or pharmaceutically acceptable salts, solvates andpolymorphs thereof in the treatment of PDE related disorders, includingpulmonary disorders e.g: asthma, bronchitis, chronic obstructivepulmonary disease (COPD), pulmonary arterial hypertension; allergicdiseases e.g.: allergic rhinitis and allergic conjunctivitis; endotoxinshock; inflammatory bowel diseases e.g. ulcerative colitis and Crohn'sdisease; joint disorders such as arthritis, psoriatic arthritis andosteoarthritis; coronary heart disease; intermittent claudication;mental disorders e.g.: dementia, depression, schizophrenia; erectiledysfunction; Duchenne muscular dystrophy; male and female fertility; Dryeye disorder, psoriasis, rosacea, dermatitis; fibrotic skin diseasese.g. keloids, hypertrophic scarring, collagenoma and scleroderma;Alopecia areata, Lichen Planus, Pemphigus foliaceus, Pemphigus vulgaris,chronic periodontitis, Dermatitis Herpetiformis, Vitiligo and BullousPemphigoid. The compounds of Formula-I have been found to treat PDErelated disorders by inhibiting PDEs and various downstream mechanisms,including, but not limited to, inhibiting release of cytokines andchemokines; inhibiting release of one or many markers of inflammationsuch as TNF-α, STAT3, NFκB, and combinations thereof.

The anti-parasitic drugs of Formula-I such as Niclosamide, Oxyclozanide,Rafoxanide, Closantel, Dibromsalan, Metabromsalan, Tribromsalan andNitazoxanide, their prodrugs, their metabolites, and salts, solvates andpolymorphs thereof intended for PDE related disorders are administeredin a physiologically acceptable carrier to a subject. The treatment ofsome of the PDE related disorders may be administered in a variety ofways including but not limited to oral administration, ophthalmicadministration, nasal administration, parenteral administration,including topical, local dermal as well as transdermal, subcutaneous(s.c.), subdural, intravenous (i.v.), intramuscular (i.m.), intradermalintrathecal, intraperitoneal (i.p.), intracerebral, intra-arterial, orintralesional routes of administration, localized (e.g., surgicalapplication or surgical suppository), and pulmonary (e.g., aerosols,inhalation, or powder) and as described further below.

The correct dosage of a pharmaceutical composition comprisinganti-parasitic drugs of Formula I will vary according to thepharmaceutical formulation, the mode of application, as well as theparticular site, host and PDE related disorder being treated. Otherfactors including age, body weight, sex, diet, time of administration,rate of excretion, condition of the host, drug combinations, reactionsensitivities and severity of the disease may be readily taken intoaccount by a treating professional or one of skill in the art. Thedosage of the claimed compounds depends on several factors, including:the administration method, the disease to be treated, the severity ofthe disease, whether the disease is to be treated or prevented.

Administration may be carried out continuously or periodically withinthe maximum tolerated dose. The administration may be conducted, forexample, hourly, once every two hours, once every three hours, onceevery six hours, once every eight hours, once every twelve hours, daily,alternate day, weekly, every two weeks, every three weeks, or monthly,as needed. The dose may be administered from one day to 12 monthsdepending on disease or condition to be treated or prevented.

The topical route of administration is a preferred route for treatmentof skin diseases like psoriasis, rosacea, dermatitis and fibroticdiseases such as keloids, hypertrophic scarring, collagenoma andscleroderma as well as diseases like Alopecia areata, Lichen Planus,Pemphigus foliaceus, Pemphigus vulgaris, chronic periodontitis,Dermatitis Herpetiformis, Vitiligo and Bullous Pemphigoid. The topicaldermal composition comprising anti-parasitic drugs of Formula I actslocally and penetrate to skin layers like stratum corneum, other partsof epidermis and parts of dermis. The topical pharmaceutical compositioncomprising anti-parasitic drugs of Formula I exclude water resistant,anti-penetrant topical formulation. Suitable compositions for topicaladministration may include creams, gels, lotions, soaps, shampoos,aerosol, balm, serum, mousse, patch, paste, pump spray, roll-on, topicalsolution, stick, towelette, ointment, wipe, emulsion, cosmetic, and anycombination thereof. In the treatment of skin diseases, compounds ofFormula I of present invention have been found to be useful. Thus, thepresent invention provides a pharmaceutical composition for topicaladministration, for the treatment of skin diseases like psoriasis,rosacea, dermatitis and fibrotic skin diseases as well as diseases likeAlopecia areata, Lichen Planus, Pemphigus foliaceus, Pemphigus vulgaris,chronic periodontitis, Dermatitis Herpetiformis, Vitiligo and BullousPemphigoid, comprising anti-parasitic drugs of present invention,prodrugs thereof, or a pharmaceutically acceptable salt or solvatesthereof, in a pharmaceutically acceptable topical carrier, vehicle, ordiluent. The topical composition comprising compound of Formula-I ofpresent invention is preferably in the form of a gel, a patch, topicalsolution, cream, ointment, topical swab, emulsion, shampoo, spray orlotion. The composition may be provided in sustained release form.

Depending upon the manner of administration, the anti-parasitic drugs ofFormula-I of present invention, their prodrugs or metabolites, orpharmaceutically acceptable salts, solvates and polymorphs may beformulated in various ways. The concentration of the compound ofFormula-I in a formulation for topical administration may vary from aconcentration of about 0.001% to 20.0% w/w administered one to ten timesdaily for one week to 12 months is usually preferable. Preferably, theconcentration of the anti-parasitic drugs in a formulation for topicaladministration may vary from a concentration of about 0.001% to 10.0%w/w. In treating skin diseases like psoriasis, rosacea, dermatitis andfibrotic skin diseases, a composition containing compound of Formula-Iof present invention as the active ingredient may be advantageouslyadministered to subject in need by way of a topical preparation, havinga concentration of anti-parasitic drugs of present invention of about0.001% to 10.0% w/w in a suitable pharmaceutical carrier. In treatingpulmonary diseases, the anti-parasitic drug may be administered to amammal as an inhaler, nebulizer or vaporizer in a concentration of about0.001% to 10.0% w/w and preferably of 0.1% to 5% w/w for one to tentimes a day for one day to 12 months or more.

When administered by oral route, the dosage of the anti-parasitic drugof present invention is preferably about 0.1 mg to 3000 mg daily(desirably about 0.5 mg to 2000 mg) one to ten times daily (preferablyone to five times daily, more desirably one to three times daily) forperiod of one day to 12 months or more. When administered by ophthalmicroute, the dosage of the anti-parasitic drug of present invention ispreferably at a concentration of 0.001% to 10.0% w/v administered one toten drops i.e. 0.05 mL to 0.5 mL (preferably one to three drops i.e.0.05 mL to 0.15 mL) at each time up to ten times a day (preferably oneto five times daily, more desirably one to three times daily) for periodof one day to 12 months or more. When administered by a topical route,the dosage of the anti-parasitic drug of present invention is preferablyat a concentration of 0.001% to 20.0% w/w applied one to ten times a dayfor period of one week to 12 months or more in a quantity that issufficient to cover the affected area by the formulation with a layer ofapproximately 0.1 mm to 5 mm thickness. When administered by intravenousor intramuscular route, the concentration of therapeutically activeingredient of anti-parasitic drug of present invention is preferablyabout 0.001% to 10.0% w/v administered one to ten times a day withmaximum dose of 500 mg per application for period of one day to 12months or more.

Preferably, the anti-parasitic drugs of present invention, theirprodrugs, or pharmaceutically acceptable salts, solvates and polymorphs,the concentration of such therapeutically active ingredient in aformulation for oral administration may vary from a concentration ofabout 1 mg to 3000 mg daily for any of the PDE-related disorders. Theconcentration of the anti-parasitic drugs of present invention in aformulation for ophthalmic administration may vary from a concentrationof about 0.001% to 10.0% w/v. The concentration of therapeuticallyactive ingredient of anti-parasitic drugs of present invention in aformulation for intravenous, intradermal, intralesional or intramuscularadministration may vary from a concentration of about 0.001% to 10.0%w/v.

For parenteral administration, anti-parasitic drugs of presentinvention, their prodrugs, or pharmaceutically acceptable salts,solvates and polymorphs of the invention can be administered asinjectable dosages of a solution or suspension of the substance in aphysiologically acceptable diluent with a pharmaceutical carrier, whichcan be a sterile liquid such as water and oils with or without theaddition of a surfactant. Other acceptable diluents include oils ofanimal, vegetable, or synthetic origin, for example, peanut oil, soybeanoil, and mineral oil. In general, glycols such as propylene glycol orpolyethylene glycol (PEG) are preferred liquid carriers, particularlyfor injectable solutions. The anti-parasitic drugs of present invention,their prodrugs, or pharmaceutically acceptable salts, solvates andpolymorphs of this disclosure can be administered in the form of a depotinjection or implant preparation, which can be formulated in such amanner as to permit a controlled or sustained release of the activeingredient(s).

Pharmaceutical compositions comprising anti-parasitic drugs of presentinvention, their prodrugs, or pharmaceutically acceptable salts,solvates and polymorphs may also include pharmaceutically acceptable,non-toxic carriers or diluents, which are vehicles commonly used toformulate pharmaceutical compositions for animal or humanadministration. The formulations may also contain conventionaladditives, such as solubilizers, isotonic agents, suspending agents,emulsifying agents, stabilizers, penetrating agents, humectant andpreservatives.

The compositions may be formulated for sustained release. Theanti-parasitic drugs of present invention, their prodrugs, orpharmaceutically acceptable salts, solvates and polymorphs of thisinvention can be administered in a sustained release form, for example adepot injection, implant preparation, or osmotic pump, which can beformulated in such a manner as to permit a sustained release of theactive ingredient. Implants for sustained release formulations arewell-known in the art. Implants are formulated as microspheres, slabs,etc. with biodegradable or non-biodegradable polymers. For example,polymers of lactic acid and/or glycolic acid form an erodible polymerthat are well-tolerated by the host.

Example 1: Computational Model(s) Prediction of Anti-Parasitic DrugNiclosamide to be a Potent Phosphodiesterase Inhibitor

The screening of small molecule database using in-silico dockingtechniques suggested drug Niclosamide to be a potent candidate for PDEinhibition. GRIP docking of Niclosamide along with ligand flexibilitywith the aim of exploring the conformational space of the molecule, wasdone in various PDE isotype crystal structures, as provided in VlifeMDSsoftware (VLifeMDS version 4.1: Molecular Design Suite developed byNovaLead Pharma Pvt. Ltd., Pune, India 2014. In PDE4B (1RO6), dockingpose confirms the possibility of Niclosamide binding tophosphodiesterase mainly by two hydrogen bonds with Thr407, Gln443 andpi-stacking interactions with Phe446 and Phe414. These interactions arecommon with many of the co-crystallized ligands for PDE4B structures.Similarly, Niclosamide docked in to various crystal structures of PDE4Dand PDE10A showed promising binding in the corresponding active site.Thus, Niclosamide has shown ability to bind to one or more isotypes ofPDEs and hence can be promising PDE inhibitor. The other anti-parasiticdrugs of Formula-I have structural similarity (similarity index) withNiclosamide as provided below in Table I:

TABLE I Similarity index of drugs w.r.t Niclosamide Drug Name SimilarityIndex Dibromsalan 0.68 Tribromsalan 0.67 Metabromsalan 0.50 Rafoxanide0.75 Oxyclozanide 0.76 Closantel 0.70 Nitazoxanide 0.52 Niclosamide 1.0

The high structural similarity indicates that these drugs (compounds ofFormula-I) bind to one or more isoenzymes of PDE in a similar fashion tothat of Niclosamide and hence also are PDE inhibitors.

All other anti-parasitic drugs of Formula-I such as Nitazoxanide,Oxyclozanide, Rafoxanide, Dibromsalan, Metabromsalan, and Tribromsalanwere docked in various crystal structures of PDE isoenzymes. These drugsare found to bind effectively to one or more PDE isoenzyme structures.The information about a representative enzyme interacting withanti-parasitic drugs of present invention along with their keyinteractions similar to those with corresponding co-crystallized ligandsis summarized below in the Table-II below.

TABLE II Docking interactions of drugs with PDEs PDE PDB H-bonding Druginvolved type code residues Pi-stacking residues Niclosamide PDE4B 1RO6Thr407, Gln443 Phe446, Phe414 PDE4D 3SL4 Gln369 Phe372, Phe340 PDE10A4DFF Gln726 Phe729 Nitazoxanide PDE4B 1XM6 Gln443 Phe446, Phe414, Tyr233PDE4A 3I8V Gln581, Tyr371 Phe584, Tyr371, His372, His376 PDE10A 4AELGln726 Phe729, Phe696 Dibromsalan PDE4D 1Y2C Gln369 Phe372, Phe340PDE10A 3UUO Gln726 Phe729, Phe696 Oxyclozanide PDE4A 3I8V Tyr371 Phe584PDE10A 3UUO Gln726 Phe729, Phe696, Tyr524 PDE5 2H44 Gln817 Phe820,Phe786, Tyr612 Rafoxanide PDE7A 1ZKL Asn260 Phe416 PDE10A 4AEL Gln726Phe729, Phe696 Metabromsalan PDE4B 1RO6 Gln443 Phe446, Phe414 PDE4A 2QYKGln581 Phe584, Tyr371, Phe552 Tribromsalan PDE5A 3TGE Gln817 Phe820,Phe786, Tyr612 PDE4B 1RO6 Gln443 Phe446 Closantel PDE4A 3I8V His372Tyr371, Phe584 PDE7A 1ZKL Asn260 Tyr419 PDE4D 3SL4 Asn209, Gln343,Phe372 Ser355

The above results indicated that anti-parasitic drugs of presentinvention (compounds of Formula-I) have potential to bind and inhibitone or more types of PDE isomers.

Example 2: Evaluation of Phosphodiesterase Inhibition Action ofNiclosamide and Nitazoxanide in Radiolabeled Enzyme Assay

The following enzymes were utilized to check PDE inhibitory activity ofNiclosamide and Nitazoxanide: PDE1 (Bovine brain), PDE2 (Humanplatelets), PDE3 (Human platelets), PDE4 (Human U937 cells), PDE5 (Humanplatelets). Enzyme assays have been optimized for incubation time,substrate concentration and method of end-product determination. Thesubstrate used for PDE1 to PDE4 are 1.01 μM [³H] cAMP+cAMP; whereas forPDE5 it is 1.01 μM [³H] cGMP+cGMP.

The results of % inhibition of various isotypes of PDE by Niclosamideand Nitazoxanide are given below in Table III:

TABLE III PDE inhibition of Niclosamide and Nitazoxanide in radiolabeledenzyme assay S. Phosphodiesterase % inhibition at 30 μM % inhibition at30 μM No. Type of Niclosamide of Nitazoxanide 1 PDE1 61 18 2 PDE2 16 133 PDE3 62 −4 4 PDE4 27 89 5 PDE5 49 9

The above data shows that Niclosamide has good ability to inhibit manyPDE isotypes. The binding of Niclosamide with PDE isotypes was furtherconfirmed in experiments and detailed studies. IC50 values ofNiclosamide were determined for PDE3 and PDE1, which were found to be21.3 μM and 72.9 μM respectively. The data shows that Niclosamide hasinhibitory activity against multiple PDEs. The above data also showsthat Nitazoxanide has PDE4 inhibitory activity.

Example 3: In-Vitro Evaluation of PDE Inhibition of Drugs byFluorescence Polarization Assay

An experiment to check inhibition of various subtypes of the targetPhosphodiesterase (PDE) enzyme in in-vitro assay was performed. Thein-vitro experiment determined the IC50 values of Niclosamide,Nitazoxanide and Tizoxanide in 10 PDE subtypes viz. PDE1A, PDE2A, PDE4A,PDE4B, PDE4C, PDE4D, PDE5A, PDE7A, PDE9A, PDE10A.

The result of IC50 of three drugs against various PDE subtypes is givenin Table IV

TABLE IV IC50 values of Niclosamide, Nitazoxanide and Tizoxanide for PDEsubtypes. PDE IC50 (μM) Enzyme Substrate Niclosamide NitazoxanideTizoxanide PDE1A cGMP, Ca-CaM 88.66 137 96.1 PDE2A cGMP 11.59 67.5 12.0PDE4A cAMP 100.00 — — PDE4B cAMP 69.06 >500 >500 PDE4C cAMP 20.22 —366.0 PDE4D cAMP 111.20 — — PDE5A cGMP 16.70 — 136.0 PDE7A cAMP 19.12 —134.0 PDE9A cGMP 59.82 — — PDE10A cAMP 19.05 — —

The data shows that Niclosamide, Nitazoxanide and Tizoxanide inhibit oneor more PDE subtypes. Niclosamide is a non-selective inhibitor of PDEwhereas Nitazoxanide and its metabolite Tizoxanide show inhibition ofPDE2A and PDE A.

Example 4: In-Vitro Evaluation of Inhibition of Keratinocyte (HaCat)Proliferation

An experiment to check keratinocyte proliferation inhibitory activity ofselected compounds of Formula-I was performed to establish itsapplication in skin disorders related to keratinocytehyper-proliferation. HaCaT cells, a human immortalized cell line derivedfrom keratinocytes, representing hyper-proliferating keratinocytes wasinvestigated for anti-proliferative potential of the selected compoundsby MTT assay.

The results of % Cell proliferation w.r.t. control for Niclosamide,Nitazoxanide and Tizoxanide at 24 h, 48 h and 72 h are given in Table V:

TABLE V Inhibition of cell proliferation w.r.t. control at 24, 48 and 72hrs for Niclosamide, Nitazoxanide and Tizoxanide Cell proliferation (%of Control) Compound Concentration μM 24 h 48 h 72 h Niclosamide Control100 100 100 0.01 129.5 124.8 109.5 0.1 114.5 103.8 94 0.5 121.5 70.566.5 1 94.8 55 60.4 2.5 94.8 57.9 55 5 96.7 62 40.7 10 107.4 64.1 39.225 115.4 74.6 37.6 IC₅₀ NA NA  3.25 uM Nitazoxanide Control 100 100 1000.01 126.3 113.7 114.6 0.1 114.3 115.2 102.4 0.5 120.3 113.4 106.2 1 123110.8 101.7 5 121.6 91.8 82.9 10 103.8 77 69.5 50 92.6 69.6 49.6 10089.7 61 33.1 IC₅₀ NA NA 39.94 uM Tizoxanide Control 100 100 100 0.0198.2 109.5 87.4 0.1 93.9 120.1 82.7 0.5 93.2 108.9 87.7 1 99.7 112.4 815 101.6 89.3 77.9 10 94.9 74.1 64.3 50 67.3 59.9 38 100 53.5 38.6 22.8IC₅₀ NA 59.89 uM 22.35 uM

The above data shows that Niclosamide, Nitazoxanide and Tizoxanide havegood ability to inhibit keratinocyte proliferation after 48 Hrs. Thisclearly indicates that compounds of Formula-I of present invention havea promise in one or more skin diseases involving keratinocytehyper-proliferation.

Example 5: In-Vitro Evaluation of Drugs on Cell Viability and CytokineProduction in Human PBMCs

Fresh human blood from healthy volunteer was collected in anticoagulant(5 mM) ethylene diamine tetra acetic acid (EDTA) containing tubes. Thegrowth medium used was RPMI-1640 medium (with 3% FBS) and incubated at37′C for 60 mins in a 5% CO2 incubator with vehicle used as 1% DMSO. Thecell viability was checked at various time points using Alamar blue andfluorescence readings were taken at excitation of 544 nm and emission of590 nm.

Results:

The results of % Cell viability w.r.t. control for Niclosamide,Nitazoxanide and Tizoxanideat 24 h, 48 h and 72 h are given in Table VI:

TABLE VI % Cell viability of PBMCs in presence of drugs at variousconcentrations and at various time points Concentration % Viability(±S.E.M.) Compound (μM) 24 hrs 48 hrs 72 hrs DMSO control — 100 ± 0  100± 1  100 ± 4  Dexamethasone 3 82 ± 6 78 ± 1 81 ± 1 Doxorubicin 3 110 ±1  70 ± 5 52 ± 1 Niclosamide 100 27 ± 5  2 ± 1 −2 ± 0  1 94 ± 4 63 ± 060 ± 1 0.01 134 ± 7  107 ± 1  107 ± 6  Nitazoxanide 100 76 ± 4 42 ± 4 11± 3 1 91 ± 2 105 ± 7  106 ± 2  0.01 97 ± 1 106 ± 8  113 ± 10 Tizoxanide100 91 ± 5 64 ± 2 25 ± 2 1 84 ± 2 101 ± 4  102 ± 3  0.01 91 ± 3 96 ± 3115 ± 1 

The concentration of selected compounds of Formula-I, which exhibitedcell viability of 70% was considered as maximum concentration at whichthe stimulation of PBMCs should be studied for the respective compound.This was found to be 2.5 μM for Niclosamide and 50 uM for Nitazoxanideand Tizoxanide, hence concentrations below this concentration were usedto study various cytokine inhibition/induction upon stimulation ofPBMCs. All measurements were done in triplicate.

Stimulants Used:

PBMCs were stimulated with different stimulators as follows:

-   -   a. Cells were exposed to stimulator LPS at 100 ng/mL for 18 hrs        to stimulate TNF-α, IL-6, IFN-γ, IL10, IL-1β    -   b. Cells were exposed to stimulator IFN-γ+LPS at 100 ng/mL+1        μg/mL for 18 hrs to stimulate IL-12    -   c. Cells were exposed to stimulator PMA+Ionomycin at 50 ng/mL+1        μg/mL for 18 hrs to stimulate IL-23, IL-22, IL-17A, IL-17F

After 18 hrs supernatants were collected and centrifuged to remove anycell debris and aliquots were used for ELISA.

Results

Effect of selected compounds of Formula-I on induction/inhibition ofinflammatory cytokines in stimulated PBMCs is shown below.

TABLE VII Cytokine concentration in (pg/mL) in presence of stimulus andvarious drug concentrations Conc Human Human Human Human Human HumanHuman Human Human (μM) IL-22 IL-17A IL-17F TNF-α IL-6 IL-10 IL-12 IFN-yIL-1β Cells + —  0 ± 0 0 ± 0 0 ± 0  65 ± 3 1471 ± 47 128 ± 0 114 ± 0 178± 13  903 ± 30 0.5% DMSO Cells + — 9421 ± 848 381 ± 0  364 ± 16   3296 ±193 5568 ± 781 1222 ± 47  4351 ± 237 204 ± 13 2138 ± 99 0.5% DMSO +Stimulus* Dexamethasone 1 2496 ± 34  186 ± 0  322 ± 10  325 ± 7 3052 ±221  710 ± 21 163 ± 0 165 ± 0  174 ± 0 Niclosamide 2.5 84 ± 0 0 ± 0 0 ±0 3074 ± 91 3103 ± 75  53 ± 0 138 ± 0 165 ± 26 2166 ± 31 1 756 ± 16 0 ±0 0 ± 0 2938 ± 15 4435 ± 245  89 ± 13  663 ± 12 125 ± 14 4519 ± 1373 0.52795 ± 195 0 ± 0 0 ± 0 3839 ± 73 5653 ± 697  837 ± 65 1359 ± 0  125 ± 144126 ± 596 0.1 10574 ± 0   350 ± 32  202 ± 2   4611 ± 150 5682 ± 668 1645 ± 180 1221 ± 0  165 ± 26 4287 ± 959 Nitazoxanide 50  0 ± 0 0 ± 0 0± 0  2896 ± 117 2531 ± 94  42 ± 11  101 ± 12 191 ± 0 2506 ± 139 25 228 ±0  0 ± 0 0 ± 0  3679 ± 123 3567 ± 127  76 ± 0 406 ± 0 152 ± 13 3203 ±326 10 1093 ± 6  0 ± 0 0 ± 0 3472 ± 50 5342 ± 386  512 ± 19 1500 ± 29125 ± 14 3971 ± 217 1 5612 ± 373 39 ± 0  166 ± 10  5708 ± 27 5520 ± 5061799 ± 79 3892 ± 88 217 ± 0 4470 ± 636 Tizoxanide 50  0 ± 0 0 ± 0 0 ± 02440 ± 13 2086 ± 38  42 ± 11  101 ± 12 191 ± 0 2522 ± 80 25 280 ± 0  0 ±0 0 ± 0 3489 ± 34 3428 ± 154  76 ± 0 381 ± 0 139 ± 0 4168 ± 1079 10 1189± 18  0 ± 0 0 ± 0 3244 ± 48 5225 ± 649 493 ± 0 4101 ± 42 111 ± 0 4118 ±853 1  7858 ± 1361 39 ± 0  151 ± 5   5351 ± 174 6122 ± 865 1591 ± 25 4715 ± 176 204 ± 13 4577 ± 975 *Stimulus and its concentration for eachcytokine release as per information above* Stimulus and its concentration for each cytokine release as perinformation above Inferences drawn from above table are:1. Cytokine assays stimulated with LPS or LPS+ IFN-γ: All the testedcompounds appear to show robust and dose dependent inhibition of IL-12and IL-10, moderate inhibition of IL-6, little or no effect on TNF-α,IFN-γ, and IL-1β.2. Cytokine assays stimulated with PMA+Ionomycin: All the testedcompounds appear to show robust inhibition of Th17-type cytokinesIL-17A, IL-17F and IL-22. PMA+Ionomycin did not induce IL-23.

Example 6: In-Vitro Evaluation of Drugs on Cell Viability and CytokineProduction in HaCaT Cells

HaCaT cells were maintained in T-75 cm2 flask in Dulbecco's modifiedEagle medium supplemented with FBS (10%), L-glutamine (2 mM), penicillin(100 ug/ml), streptomycin (100 ug/ml) at 37° C. in 5% CO2 incubator.Cell growth was maintained to 70-80% confluence for seeding and vehicleused was 1% DMSO. The cell viability was checked at various time pointsusing Alamar blue and fluorescence readings were taken at excitation of544 nm and emission of 590 nm.

Results:

The results of % Cell viability w.r.t. control for Niclosamide,Nitazoxanide and Tizoxanideat 24 h, 48 h and 72 h are given in TableVIII:

TABLE VIII % Cell viability of HaCaT in presence of drugs at variousconcentrations and at various time points % Viability (± S.E.M.)Compound Conc (μM) 24 hrs 48 hrs 72 hrs DMSO control — 100 ± 11 100 ± 2 100 ± 2  Dexamethasone 3 46 ± 3 18 ± 2  8 ± 0 Doxorubicin 3 74 ± 6 108 ±5  110 ± 2  Niclosamide 50 28 ± 1 16 ± 0 23 ± 7 10 38 ± 2 29 ± 1 33 ± 25 48 ± 1 36 ± 2 45 ± 8 1 62 ± 4 46 ± 0  59 ± 11 0.1 111 ± 9  82 ± 3 98 ±6 Nitazoxanide 100 17 ± 1 13 ± 1 17 ± 1 50 18 ± 1 15 ± 2 18 ± 1 10 41 ±1 40 ± 2 46 ± 3 1 81 ± 3 86 ± 8 91 ± 2 0.1 89 ± 1 99 ± 9 103 ± 3 Tizoxanide 100 20 ± 0 13 ± 0 20 ± 0 50 24 ± 1 20 ± 0 25 ± 0 10 34 ± 1 35± 0 43 ± 1 1 74 ± 6 91 ± 5 91 ± 2 0.1 76 ± 6 114 ± 2  112 ± 6 

The concentration of compounds of Formula-I which exhibited cellviability of 70% was considered as maximum concentration at which thestimulation of HaCaT cells should be studied. This was found to be 0.25μM for Niclosamide, 1 uM for Nitazoxanide and Tizoxanide, and henceconcentrations below this concentration were used to study variouscytokine inhibition/induction upon stimulation of HaCaT cells. Allmeasurements were done in triplicate.

Stimulants Used:

HaCaT cells were stimulated with different stimulators as follows:

-   -   a. Cells were exposed to stimulator LPS at 1 μg/mL for 48 hrs to        stimulate IL-1β    -   b. Cells were exposed to stimulator IFN-γ at 50 ng/mL for 48 hrs        to stimulate TNF-α, IL-6

After 48 hrs supernatants were collected and centrifuged to remove anycell debris and aliquots were used for ELISA.

Results

Effect of compounds of Formula-I on induction/inhibition of inflammatorycytokines in stimulated HaCaT cells is shown in Table IX.

TABLE IX Cytokine concentration in (pg/mL) in presence of stimulus andvarious drug concentrations Conc. TNF-α IL-6 Group (μM) Conc. (pg/mL)DMSO 0.50% 2.8 ± 0.1 17 ± 1 Control (0.5% DMSO + Stimulus) 3.3 ± 0.1 82± 1 Dexamethasone 1 3.2 ± 0   33 ± 1 Niclosamide + Stimulus 0.25 2.5 ±0.1  54 ± 11 0.1 2.8 ± 0.1 76 ± 4 0.05 2.5 ± 0.1 103 ± 4  Nitazoxanide +Stimulus 1 2.3 ± 0.4 70 ± 4 0.5 2.5 ± 0.5 79 ± 4 0.1   3 ± 0.2 83 ± 1Tizoxanide + Stimulus 1 2.3 ± 0.5 78 ± 6 0.5 3.2 ± 0.1 80 ± 2 0.1 2.9 ±0.2 87 ± 2

It can be seen from above table that HaCaT cells produced moderatelevels of IL-6, very low levels of TNF-α and no IL-1β in response toIFN-γ. Reduction in IL-6 release was observed in HaCaT cells treatedwith 0.25 μM of Niclosamide.

Example 7: Topical and Transdermal Formulations of Antiparasitic Drugs

TABLE X Compositions of topical and transdermal formulationsIngredient/Excipient NIC-1 NIC-2 NIT-1 NIT-2 Niclosamide 3% 3% — —Nitazoxanide — — 5% 5% Petrolatum 96%  81% 94%  79%  Lanolin 1% 1%Methyltriglycol 5% 4.5%   Liquid paraffin 10% 10.5%   Sorbitansesquioleate 1% 1%

The compositions NIC-1 and NIT-1 are topical dermal whereas NIC-2 andNIT-2 are transdermal compositions.

Example 8: In-Vivo Evaluation of Drugs by Imiquimod Induced PsoriasisModel in Balb/c Mice

In-vivo experiment to check anti-psoriatic potential of Niclosamide andNitazoxanide was performed in Mus Musculus species. BALB/c mice (Sex:Female, Age: 7-8 Weeks) were used for induction of psoriasis using IMQ.Topical application of imiquimod (IMQ) in mice can induce and exacerbatepsoriasis closely resembling human psoriasis. Concurrent treatment withvarious concentrations of Niclosamide and Nitazoxanide were investigatedto quantify the reduction in parameters of psoriasis in the IMQ inducedmice. Nine groups (8 animals per group) were used including normalcontrol, disease control; three concentrations of Niclosamide, viz 1%,2% and 3%; three concentrations of Nitazoxanide, viz. 3%, 4% and 5% andpositive control of Tenovate (Clobetasol) 0.05% cream. In all 63 mgcream containing 5% of IMQ was applied to dorsal area (shaved back) andright ear for 8 days. Topical formulations from Example 7 were usedduring this experiment. Three hours after IMQ application, differentdoses of Niclosamide (1%, 2% and 3%) and Nitazoxanide (3%, 4% and 5%)were applied to dorsal area (50 mg) and ear (25 mg) in the correspondinggroups. The Niclosamide, Nitazoxanide or vehicle doses were appliedtwice daily.

The parameters investigated were ear thickness change, % inhibition ofear inflammation, Psoriatic Area Severity Index (PASI). Histopathologywas performed of right ear and dorsal skin by H & E staining. Thebiomarker in this study was inflammatory cytokine IL-23 analyzed byELISA obtained from tissue lysate of ear region where IMQ and doses wereapplied.

The results of in-vivo studies for Niclosamide and Nitazoxanide aregiven in table X:

TABLE XI Parameters evaluated in IMQ induced psoriasis assay forNiclosamide and Nitazoxanide. % % Body Spleen inhibition IL23Histopathology Scores Weight weight of Ear PASI Levels Epidermal RetePeg Group Change (mg) inflamn. score (pg/ml) Hyperkeratosis ThicknessProlifern. Normal Control 10.8 141.6 ± 15.5 100.0 0.00 83.9 ± 6.8 1.00 ±0.0  4.13 ± 0.1  0.00 Disease Control −1.9 222.5 ± 19.3 0.0 20.3 ± 1.6249.5 ± 52.0 1.7 ± 0.2 6.7 ± 0.2 1.7 ± 0.2 Niclosamide 1% −6.0 265.2 ±49.2 27.1 15.8 ± 3.0 119.9 ± 7.8*  0.8 ± 0.2^(#) 6.8 ± 0.2 1.8 ± 0.2Niclosamide 2% −7.5 214.4 ± 15.1 30.4 17.2 ± 1.7  96.6 ± 10.9* 1.4 ± 0.25.6 ± 0.8 1.4 ± 0.2 Niclosamide 3% −6.0 229.7 ± 18.7 42.7  10.7 ± 4.1* 83.6 ± 9.8*  1.0 ± 0.0^($) 5.2 ± 0.2  0.5 ± 0.2^(#) Nitazoxanide 3%−4.75 203.6 ± 22.6 −5.9  14.2 ± 5.1^(#) 147.79^(#)  1.0 ± 0.0^($) 5.6 ±0.4 1.2 ± 0.2 Nitazoxanide 4% −0.59 274.7 ± 54.3 26.1 21.5 ± 3.4 128.67*1.3 ± 0.2  4.7 ± 0.3^(#) 1.0 ± 0.3 Nitazoxanide 5% −3.78 204.1 ± 15.340.9  13.1 ± 1.6* 89.45* 1.1 ± 0.1 5.3 ± 0.6 1.1 ± 0.1 Clobetasol 0.05%−16.9  49.3 ± 10.3 86.9 4.83 ± 2.3 64.1 ± 7.4  1.0 ± 0.0*  4.3 ± 0.3^(#) 0.3 ± 0.3^(#) *p < 0.001, ^(#)p < 0.01, ^($)p < 0.05; one Way ANOVAfollowed by Dunett's test, compared to Disease control

The above results from Table XI show that both Niclosamide andNitazoxanide have good ability to inhibit proliferation of skinthickening and reduce inflammatory processes in disease, thusdemonstrating anti-psoriatic potential of the drugs. The tested drugsshowed dose dependent reduction in ear inflammation at Day 7.Niclosamide and Nitazoxanide inhibited 42.7% and 40.9% of the earinflammation respectively at highest tested concentrations. Psoriaticarea severity index (PASI) scores in the treatment groups showsignificant reduction in severity of disease. Niclosamide showed up to50%/a reduction in the PASI score at 3% concentration indicatinganti-psoriatic potential. A significant increase was observed in IL-23levels (p<0.001) on Imiquimod application in ear tissue homogenates. Theanimals treated with the Niclosamide and Nitazoxanide at different dosesshowed significant (p<0.01, p<0.001 respectively) reduction of IL-23level. Niclosamide treatment showed dose dependent response where in thehighest concentration reduced the IL23 levels to normal controlsuggesting ability of the compound to reduce inflammation. On the otherhand, Clobetasol reduces IL-23 levels below that of normal controlindicating that this may hamper safety of the drug. The inhibition ofIL-23 by compounds of Formula-I leads to anti-inflammatory action seenin this disease model. The concentration dependent reduction of cytokineIL-23 by both Niclosamide and Nitazoxanide indicate their potential intreatment of diseases associated with upregulation of IL-17/IL-23 axisincluding skin diseases like psoriasis, rosacea and eczema.

The results from Table XI demonstrated that no significant body weightchange was observed in the Niclosamide and Nitazoxanide groups; whereasClobetasol showed significant body weight change from Day 7 to Day 9(p<0.01, p<0.001). The significant reduction in body weight ofClobetasol group is an indicator of drug toxicity. On Day 9, all theexperimental animals were euthanized, spleen was collected and weighed.Among the multiple treatment groups; marginal reduction in spleen weightwas observed in treatment with Niclosamide 2%, Nitazoxanide 3% and 5%,however it was not statistically significant when compared with diseasecontrol. The reduction in spleen weight was observed to be significantin Clobetasol group indicating deleterious effects on spleen cells.

The histopathology studies of the ear lobes were performed and criticalparameters like Hyperkeratosis, Epidermal Thickness and Rete pegproliferation were recorded. The evaluation shows dose dependentreduction in Epidermal thickness and Rete peg Proliferation. Asignificant reduction in Hyperkeratosis was shown by both Niclosamideand Nitazoxanide. Thus, compounds of Formula-I such as Niclosamide andNitazoxanide exhibit anti-psoriatic potential with good safety profile.

It will be appreciated that, although specific embodiments of theinvention have been described herein for purposes of illustration,various modifications may be made without departing from the spirit andscope of the invention. All such modifications and variations areintended to be included herein within the scope of this disclosure.

The invention claimed is:
 1. A method of treating phosphodiesteraserelated disorders in a mammal, comprising administering to a mammal inneed of such treatment a therapeutically effective amount of acomposition comprising an anti-parasitic compound havingphosphodiesterase inhibitory activity; wherein the anti-parasiticcompound having phosphodiester inhibitory activity is administered to amammal by an oral route in a dosage of about 1 mg to 3000 mg daily inthe form of oral solution, suspension, syrup, dental paste or a capsule;or is administered to a mammal by an ophthalmic route in a concentrationof about 0.001% to 10.0% w/v in the form of eye drops or eye gel; or isadministered to a mammal by a topical route in a concentration of about0.001% to 20.0% w/w in the form of a cream, gel, patch, ointment,topical swab, emulsion, solution, paste, shampoo, or spray with orwithout applicator; or is administered to a mammal by an intravenous,intradermal, intralesional or subcutaneous route in a concentration ofabout 0.001% to 5.0% w/v in the form of an injection or infusion; or isadministered to a mammal as an inhaler, nebulizer, or vaporizer in aconcentration of about 0.001% to 5.0% w/w.
 2. The method of claim 1,wherein the anti-parasitic compound has the structure of Formula I

wherein R₁ is —H; R₂ is selected from —H, —Cl, —Br; R₃ is selected from—H, —Cl, —Br; R₄ is selected from —H, —Cl; Ar is

wherein R₅ is selected from —H, —NO₂, —Br; R₆ is selected from —H, —Cl;R₇ is selected from —H, —OH, —Cl; R₈ is selected from —H, —Cl; andenantiomers, diastereomers, prodrugs or metabolites thereof, orpharmaceutically acceptable salts, solvates and polymorphs thereof in apharmaceutically acceptable carrier, vehicle, or diluents.
 3. The methodof claim 2, wherein the compound of formula I is selected from the groupconsisting of Niclosamide, Oxyclozanide, Dibromsalan, Metabromsalan, andTribromsalan, and pharmaceutically acceptable salts, solvates andpolymorphs thereof.
 4. The method of claim 1, wherein thephosphodiesterase related disorder is selected from the group consistingof Asthma, Bronchitis, Chronic obstructive pulmonary disease (COPD),Pulmonary arterial hypertension, Allergic rhinitis, Allergicconjunctivitis, Dry eye disorder, Coronary heart disease, Intermittentclaudication, Dementia, Depression, Schizophrenia, Erectile dysfunction,Duchenne muscular dystrophy, Male and female fertility disorders,Psoriasis, Eczema, Rosacea, fibrotic skin disease, Alopecia areata,Lichen Planus, Pemphigus foliaceus, Pemphigus vulgaris, Chronicperiodontitis, Dermatitis Herpetiformis, Vitiligo and BullousPemphigoid.
 5. The method of claim 1, wherein the mammal is a primate,canine, feline, bovine, ovine, porcine, camelid, caprine, rodent orequine.
 6. The method of claim 4, wherein the phosphodiesterase-relateddisorder is selected from Psoriasis, Rosacea and Eczema.
 7. The methodof claim 4, wherein the phosphodiesterase related disorder is selectedfrom Alopecia areata, Lichen Planus, Pemphigus foliaceus, Pemphigusvulgaris, chronic periodontitis, Dermatitis Herpetiformis, Vitiligo,Bullous Pemphigoid, and the fibrotic skin disease is selected from thegroup consisting of Keloids, Hypertrophic scarring, Collagenoma, andScleroderma.
 8. A method of treating phosphodiesterase related disordersin a mammal comprising topical application of a composition comprisingniclosamide 3%, petrolatum 96% and lanolin 1% to a mammal in need ofsuch treatment.